Geographical distribution of MTHFR C677T gene polymorphisms among the reproductive-age women in Chinese Han populations: based on migration

Background Methylenetetrahydrofolate reductase (MTHFR) is essential for the metabolism of folic acid and homocysteine. The MTHFR C677T polymorphism is associated with several disorders. Our study aims to explore the geographical distributions of the MTHFR C677T polymorphism of women in China and how migration affected the polymorphism in Suzhou. Methods A total of 7188 women of reproductive age were recruited in Suzhou of the study. Subjects were classified according to their native places after data extraction. MTHFR C677T gene polymorphisms were detected by quantitative PCR with genomic DNA isolated from blood samples. Results The frequencies of the 677T allele and 677TT genotype were higher in northern China than that in southern China and decreased in geographical gradients from north to south. The frequencies were considerably higher in the migrant population than that in the indigenous population of Suzhou. The migrant population have gradually changed the prevalence in Suzhou. Conclusions Our study suggested that the prevalence of MTHFR C677T polymorphisms among women varied across different geographical regions in Chinese Han populations. The 677T allele frequencies of the northern populations were significantly higher than those of the southern populations. The migrant population gradually changed the prevalence of the MTHFR C677T polymorphism in Suzhou. Supplementary Information The online version contains supplementary material available at 10.1186/s12905-024-03244-3.


Background
Birth defects are a serious public health and societal problem in China.Approximately 900,000 neonates are born with birth defects annually, with an incidence of 5.6% [1].The incidence of perinatal birth defects has increased in the last decade.Premarital health care and counseling is a critical component of maternal and child health, improving pregnancy outcomes and reducing birth defects and postpartum disease rates [2,3].
Folic acid, also known as vitamin B9, must be obtained from diet and supplements, which is required for DNA replication and protein metabolism [4,5].It is now wellrecognized that the consumption of folic acid prior to conception can effectively reduce birth defects, including neural tube defects (NTDs) [3,4].The health authorities issued guidelines [6] that recommended a daily folic acid addition of 0.4 to 0.8 mg for women of childbearing age.The recent concept of personalized medicine demands that dose selection must be based on the physiological/ pathological conditions of each individual [7,8].
Methylenetetrahydrofolate reductase, encoded by the MTHFR gene, is a critical regulatory enzyme engaged in the metabolism of homocysteine and the folate cycle, and it converts folic acid to tetrahydrofolic acid [9,10].It has been confirmed that the MTHFR gene variants C677T and A1298C are associated with low enzyme activity [11].Decreased enzyme activity results in elevated intracellular homocysteine, which is implicated in increased risk for a variety of disorders, including central nervous system dysfunction, birth defects, and pregnancy complications [12,13].The C677T polymorphic site is characterized by a change of C into T at position 677 of the MTHFR gene (located 1p36.3).At codon 222, an alanine to valine substitution resulted in thermolability and reduced enzyme activity.Mutant homozygote (TT genotype) attenuates enzyme activity by 65% and heterozygote (CT genotype) by approximately 40% [14].Therefore, detecting the genotype of each individual, as well as understanding the country-wide distribution is crucial for individual risk assessment, preventive medicine, and precision medicine.
Epidemiological studies have reported that the prevalence of the MTHFR C677T polymorphism varied apparently across different geographical distributions and that the frequency of MTHFR 677T allele is higher in Europe than that in Africa.It shows an increasing trend from north to south among Europeans and North Americans [11,[15][16][17].In China, it shows a reverse trend [18], where a higher frequency was more likely detected in the northern provinces.Several studies performed among Chinese populations show distributional differences [11,19].Interestingly, the distribution of the proportion of several disorders, such as recurrent abortion, preeclampsia [20,21], chromosomal aneuploidy [22] and NTDs [23], is similar to the MTHFR C677T gene distribution in China.Higher frequency of MTHFR 677T allele correlates with increased proportion of these disorders.Taking NTDs in China as an example, the prevalence is higher in the north than in the south (4.5‰ and 1.1‰, respectively) [24].Additionally, Both the plasma folate concentrations and daily dietary folate intake of women in the north were lower than those of women in the south [25][26][27].These data indicated that there are inner associations among MTHFR C677T gene distribution, folic acid intake and several disorders.These associations are complex and delicate, arising from long-term interactions between heredity and the environment.
Previous studies on geographical distributions of gene polymorphisms usually required collection of data from a large sample of subjects across various regions, which might be laborious and time-consuming, and demanded collaboration across multiple research teams.In our study, we took advantage of the demographic characteristics of Suzhou city, where our hospital located in and the study was carried out.Suzhou is a typical city of migrants.Benefitting from the booming local economy and increasing employment opportunities, approximately half of the population migrated from other cities or provinces in recent decades.It is convenient for our laboratory to obtain biological samples of subjects from various regions across China.We detected and evaluated the prevalence of MTHFR C677T polymorphisms among women of reproductive age residing in Suzhou (including migrants from other cities or provinces) and tried to elucidate its geological distribution pattern across China.Our study showed the geographical distribution pattern in a different way, which may help the government understand the trend of changes of the prevalence and formulate dynamic risk assessments.

Study population
The subjects were randomly selected reproductive-age women who had received inpatient or outpatient medical care in the Dept. of Obstetrics & Gynecology, Suzhou Ninth People's Hospital, between July 2017 and December 2019.The inclusion criteria were as follows: (I) subjects were all native Chinese women; (II) subjects were aged 18-45 years; (III) subjects came for pre-pregnancy care or pregnancy care; (IV) subjects were healthy without basic diseases; (V) subjects could offer valid personal information not restricted to age, ethnic background, place of birth, or native place; and (V) subjects were required to have the MTHFR (C677T) gene polymorphism detected to guide folic acid addition or risk assessment.
After data extraction, a total of 7188 Han subjects were recruited from the Suzhou Ninth People's Hospital in this study (mean age was 29.9 ± 6.7 years).Inquiries of census register ground was needed to attenuate the impact of migrants.All subjects were grouped into zones according to their own native places.Additionally, subjects were stratified into binary groups, the northern populations and the southern populations.According to previous studies, the divisional line was set at the Qinling Mountains-Huaihe River.To evaluate the impact of migration, subjects were divided into an indigenous population and a migrant population.
The study was performed in accordance with the Declaration of Helsinki, and the Ethics Committee of the Suzhou Ninth People's Hospital granted ethical approval for the study (approval number: KY2022-100-01).Informed consent was acquired from all participants and subsequently anonymized.

Genotyping
MTHFR C677T gene polymorphisms were detected by quantitative PCR.Genomic DNA was extracted from blood, using the TIANamp Blood DNA Kit according to the manufacturer's instructions.(TIANGEN, CN).The assays were carried out with a TaqMan PCR Core Reagent Kit (ABI, USA).The primers and probes listed below (ABI, USA) were used [11]: for: PCR amplifications consisted of an initial step of 95 °C for 2 min and 45 cycles of 95 °C for 15 s and 60 °C for 1 min.

Statistical analysis
All statistical analyses were performed using R 4.2.0 and the level of statistical significance considered was a p value below 0.05 unless otherwise stated.The 95% CIs of allelic and genotypic frequencies were calculated using the normal approximation.χ 2 testing was performed to calculate Hardy-Weinberg equilibrium among the observed genotype frequencies.χ 2 testing was performed to test the differences in allelic and genotypic frequencies between the northern and southern populations.

Hardy-weinberg equilibrium
The genotypic distribution of the MTHFR C677T polymorphism was consistent with Hardy-Weinberg equilibrium, except for the MTHFR C677T polymorphism among the population from Henan Province.

Impact of migration on frequencies
The subjects were divided into 2 major categories (migrant and indigenous) according to their registered native places.Balance diagnostics after propensity score matching (PSM) showed that the groups were comparable and other non-treatment factors were excluded (Supplementary Table 1).We found that the total pooled frequencies of the 677T allele and 677TT genotype were significantly higher in the migrant population (47.89% and 24.34%, respectively) than that in the indigenous population (37.62% and 14.36%, respectively) (p < 2.2 × 10 -16 ) (Table 1).

Geographical distribution of the MTHFR C677T polymorphisms
The geographical distribution of the population prevalence of the MTHFR C677T polymorphisms is shown in Table 2; Fig. 1.The subjects were distributed across 18 provinces/municipalities.According to our data, the proportion of the 677T allele and the 677TT genotype varied among 18 distinct zones.It is suggested that the frequencies of the 677T allele and the 677TT genotype decreased in geographical gradients from north to south (Fig. 1), which were high in those northern provinces such as Heilongjiang, Hebei, Shanxi, Shandong, Henan and then decreased to an intermediate level of 20 − 30% in Gansu, Shannxi and Anhui (Anhui is the only southern population in this group) and were low in the remaining southern provinces/municipalities.Generally, the frequencies of the 677T allele and the 677TT genotype were significantly higher in North China than that in South China (p < 2.2 × 10 -16 ).The heterogeneity of the migrant population changed the prevalence of the MTHFR C677T polymorphism in Suzhou in decades (Table 1; Fig. 2).

Discussion
Folic acid deficiency or excess can lead to various diseases.It is necessary to supplement folic acid in a rational way so that the supplemental dosage and the level after supplementation can be controlled within a reasonable range.Measures such as folic acid supplementation or food fortification should be further introduced to improve folic acid deficiency/insufficiency in certain high-risk folic acid deficient populations (northern populations in China, pregnant women, malnutrition, chronic alcohol use, chronic hemolytic anemia, genetic variation in genes of folate metabolism) [24].
Among the populations characterized by gene variants are mainly the MTHFR C677T genotype.It is encouraged that women (without high-risk factors) considering or planning a pregnancy take an oral daily dosage of 0.4-0.8mg folic acid from pre-conception to 3-month gestation.Women with the MTHFR 677TT genotype can increase the supplementation dose or extend the pre-pregnancy supplementation time.According to the guideline for the prevention of NTDs, a supplement dosage of 0.8-1.0mg folic acid throughout the pregnancy is recommended for women with high-risk factors.Notably, hyperhomocysteinemic patients with the MTHFR 677TT genotype should take higher doses of folic acid [28].
Patterns of the distribution of MTHFR C677T polymorphism may help us understand the prevalence status and formulate public health policies.In this study, we examined the prevalence of the MTHFR C677T polymorphism among a total of 7188 reproductive-age women residing in Suzhou and a large proportion of them migrated from other provinces.The geographical distribution of the MTHFR C677T polymorphism presented a wide and gradual pattern from south to north China.
Migration is a critical contributor to the Hardy-Weinberg equilibrium and may lead to frequency deviation of gene polymorphism.According to the sixth census data and the Suzhou Statistical Bulletin on National Economic and Social Development, the migrant population (6,538,536) of Suzhou has exceeded the indigenous population (6,538,372) since 2013 [29,30].Suzhou has been the second largest migrant city in China [31].The number of newborns of the migrant population showed a rocketing trend from 2005 to 2009 and even exceeded that of the household population (46.2 thousand vs. 41.4 thousand) in 2008 [30].It was suggested that approximately half of the pregnant women in Suzhou were migrants.After data extraction, the migrant population was almost 1.65-fold of the indigenous population (4479 vs. 2709) in the study.The differences in the population between our study and census data exist partly because of the definition of the indigenous population.It is worth noting that the "indigenous population" here refers to the population of individuals who were born in Suzhou.The "migrant population" consists of individuals who migrate to Suzhou, whether they own household registration in Suzhou or not.Actually, a considerable number of individuals have received household registration in Suzhou.Our data showed the frequencies of the 677T allele and the 677TT genotype in the migrant population, suggesting that the recent migration wave has imperceptibly changed the prevalence of the MTHFR C677T polymorphism in Suzhou.The frequencies of the 677T allele and the 677TT genotype were dramatically increased due to the influx of the migrant population from those high frequency regions (northern provinces).
To explore the country-wide distribution pattern of the MTHFR C677T polymorphism, native places were identified as the subject's geological region in view of the fact that the genotype would not change after birth, and this can offset the impact of migration in the last 20 years.The spatial-temporal demographic evolution could significantly change the genetic polymorphism of a migrant city.Considering that the age of subjects was in the range of 18-45 years old, our data reflect a natural status of approximately 20 years ago.Our data indicated that the frequencies of the 677T allele and the 677TT genotype varied widely in different regions.The 677T allele frequencies of the northern populations varied between 50.00% and 63.24%, which were significantly higher than those of the southern populations (32.03-47.11%).Furthermore, according to the statistics above, southern populations are more diversified than northern populations.These findings may be due to the diverse geographical characteristics and population histories.The geography and climate separatrix between the south and north separated two vastly different regions.As shown, the frequencies of the 677T allele and the 677TT genotype display a decreasing trend along latitude from north to south.Yang et al. reported apparent geographical trends in which the frequencies changed as an inverse V pattern in latitude and longitude [11].A recent study reported a similar pattern that the frequencies of the 677T allele and the 677TT genotype had a significant north-south difference.the frequencies of T allele of Shandong, Henan and Hebei ranked at the top in China [20].The authors suggested that the frequency of T allele was consistent with the geographical distribution of pregnancy complications.
In our study, the top three frequencies are in Hebei, Heilongjiang and Henan.The frequencies of the 677T allele and the 677TT genotype peaked in Shangdong (63.24% and 37.90%, respectively) and presented a decreasing pattern from east to west in northern China.Differing from published studies, we focus more on the impact of population migration on the distribution.Our data shows a dynamic trend that the frequencies of the 677TT genotype raised gradually due to the migration.This trend has its social significance that can provide concrete information to the Government for reference.For example, now government departments in Suzhou may increase publicity and education, increase the percentage of cases tested for the MTHFR genotype and vice versa.It suggests that estimating the frequency of a region through preliminary calculations of population inflows and outflows is a feasible way.
Previous scholars have reported that the frequencies of the 677T allele and the 677TT genotype also vary among ethnic groups in China.The mean frequency of the 677T allele of the Han population was lower than that of Tujia and higher than that of Duar, Man, She and several other ethnic groups [32].In this study, 52 ethnic minority subjects from 12 other ethnic groups were recruited.Due to the widely scattered subjects and the small sample size, these subjects were not included in the study.
In addition to the MTHFR C677T gene polymorphism, there are numerous other variations giving rise to MTHFR alleles.A previous study reported 14 nonsynonymous changes, including 11 rare SNPs each with a frequency under 1% and 3 common SNPs (C677T, A1298C and G1793A), among which SNP C677T is thought to be the best-studied case [33].MTHFR A1298C is a change of A to C at position 1298, leading to a decrease in enzyme activity, which has weaker effects than the C677T polymorphism.The frequencies of the MTHFR A1298C genotype varied considerably among different populations and showed an increasing trend from north to south [11,13].MTHFR G1793A is a G/A base transition at position 1793.The allele was associated with deficient values in folic acid [34].Investigations of the distribution of MTHFR G1793A were not adequate.Overall, the frequencies of the MTHFR 1793 A allele were lower in northern than in southern China [35].
Uncertainty surrounds the causes of the geographic gradient.Publications have indicated that the polymorphism is influenced by genetic, dietary, environmental, and demographic variables.Reinventing human relocation and migration may also be responsible for the distributional pattern [11,[36][37][38].It has come to a consensus that the historical, geographical and population diversity conditions of China bring challenges for individualized plans of folic acid supplementation.In conclusion, our study indicates that the prevalence of the MTHFR C677T polymorphisms varies considerably by region between Chinese Han populations.Our study intends to present a clear picture of the MTHFR C677T polymorphism and serve as a reference for folate intake and risk assessment for several associated anomalies.Our data can help the government formulate precise health care strategies.

Conclusions
1.The total frequencies of the 677T allele and 677TT genotype were significantly higher in the migrant population than that in the indigenous population.2. The 677T allele frequencies of the northern populations were significantly higher than those of the southern populations.3. The prevalence of the MTHFR C677T polymorphism in Suzhou is being changed and influenced gradually by the demographic diversity of migrant populations.

Fig. 1
Fig. 1 Map of China showing the geographical distributions of the MTHFR C677T polymorphisms in different regions.(A) The frequencies of the 677CC genotype increased in geographical gradients from north to south.The populations of Guangdong carried the highest frequencies of 677CC genotype.(B) The frequencies of the 677TT genotype decreased in geographical gradients from north to south.A similar pattern was recorded for the frequencies of 677T allele (C).Original map of China downloaded from website of Ministry of Natural Resources of the People's Republic of China (http://bzdt.ch.mnr.gov.cn/browse.html?picId="4o28b0625501ad13015501ad2bfc0696")

Fig. 2
Fig. 2 Impact of migrant population on the prevalence of the MTHFR C677T polymorphism in Suzhou.The northern population who carried higher frequency of the 677T allele, and the southern population who carried lower frequency of the 677T allele migrated to Suzhou, which changed the prevalence of the MTHFR C677T polymorphism in Suzhou (right panel)

Table 1
Distribution of MTHFR C677T polymorphism between indigenous population and migrant population